基于基因芯片的荧光分析方法在甲状腺乳头状癌相关BRAFV600E突变高灵敏及特异性检测中的应用  

Specific and Sensitive Detection of BRAFV600E Mutations in Papillary Thyroid Carcinoma by Oligonucleotide Microarray-based Fluorescence Assay

作　　者：魏佳[1] 高嘉雪[2] 王瑶琪 王振新[2] 孟宪瑛[1] WEI Jia;GAO Jia-Xue;WANG Yao-Qi;WANG Zhen-Xin;MENG Xian-Ying(Department of Thyroid Surgery, the First Hospital of Jilin University, Changchun 130021, China;State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, China)

机构地区：[1]吉林大学白求恩第一临床医院甲状腺外科,长春130021 [2]中国科学院长春应用化学研究所电分析化学国家重点实验室,长春130022

出　　处：《分析化学》2019年第6期855-861,共7页Chinese Journal of Analytical Chemistry

基　　金：长春市科技计划项目(No.18YJ009);国家自然科学基金项目(No.21775145)资助~~

摘　　要：研发了一种基于DNA芯片(DNA microarray)的荧光分析方法,并将其用于检测甲状腺乳头癌相关的BRAFV600突变。本方法采用三链杂交体系,首先将单链DNA(ssDNA)捕获探针固定在微阵列芯片基底上,制备DNA芯片;以其为反应平台,加入ssDNA目标物与ssDNA捕获探针进行杂交;最后加入荧光分子Cy5的修饰ssDNA标记ssDNA目标物,检测荧光信号。在优化的实验条件下,本方法对BRAFV600E突变型ssDNA目标物的检测灵敏度达到亚纳摩尔级(0.25 nmol/L),且具有3个数量级的线性范围,并能够从突变型ssDNA目标物和野生型ssDNA目标物的混合物中区分出低至0.1%的BRAFV600E突变型ssDNA目标物。利用本方法对15例临床甲状腺组织样本的BRAFV600E突变水平进行了分析,实验结果与常规Sanger测序法检测结果一致,表明本方法具有良好的实用性。A DNA microarray-based fluorescence assay was developed for profiling BRAFV600 mutations through formation of three single-stranded DNA(ssDNA) sequences hybridization systems on microarray. In this assay, the target ssDNAs were recognized by correspondent capturing oligonucleotide probes on microarray, followed by hybridization with a Cy5 modified ssDNA for fluorescence detection. Under the optimal experimental conditions, the DNA microarray-based fluorescence assay achieved a limit of detection(S/N) at sub-nanomolar level for the target ssDNA with BRAFV600 E mutation, and could easily differentiate as low as 0.1% BRAFV600 E mutation sequence in the mixture of target ssDNAs. The profile results of BRAFV600 E mutation level in 15 clinical thyroid tissue samples demonstrated that the DNA microarray-based fluorescence assay had good practicability.

关 键 词：甲状腺乳头状癌 BRAFV600E突变 DNA芯片 

分 类 号：R736.1[医药卫生—肿瘤]